Pak. J. Agri. Sci., Vol. 56(4), 943-951; 2019 ISSN (Print) 0552-9034, ISSN (Online) 2076-0906 DOI:10.21162/PAKJAS/19.3948 http://www.pakjas.com.pk

DIVERSITY PROFILE OF PROTISTS FLAGELLATES ISOLATED FROM HINDGUT OF indicola WASMANN (: ) IN

Asma Ashraf1, Naveeda Akhtar Qureshi1,*, Muhammad Haseeb1, Muhammad Qasim Hayat2, Muhammad Afzal1 and Saeed-ul-Hassan Khan1

1Department of Sciences, Faculty of Biological Science, Quaid-i-Azam University Islamabad 45320, Pakistan; 2Atta-ur-Rahman School of Applied Biosciences (ASAB), National University of Sciences and Technology (NUST), Islamabad, Pakistan *Corresponding author’s e-mail: [email protected]

Termites cause a serious menace to wood structures all over the world. They rely mostly on the entozoic fauna for the digestion of cellulosic materials. The present study is based upon the diversity of flagellates protists isolated from the gut of a lower , Heterotermes indicola, belonging to three genera i.e. Holomastigotes (H. campanula, H. annandalei and H. metchnikowi), Holomastigotoides (H. hemigynum, H. hartmanni, H. kempi, H. koidzumi and H. metchnikowi) and Pseudotrichonympha (P. grassii). The largest and most abundant species Pseudotrichonympha grassii was identified by molecular studies using the SSU rRNA gene, confirmed by phylogenetic analysis and compared with that of the P. grassii isolates reported from other parts of the world. The results showed that the P. grassii observed in our study was phylogenetically most closely related to the Japanese P. grassii isolate. The biodiversity of the entozoic flagellates is important in targeting for biological control of as well as for isolation and culturing of flagellates to produce cellulases, an important industrial enzyme. Keywords: Biodiversity, Heterotermes indicola, entozoic flagellates, Holomastigotes, Holomastigotoides, Pseudotrichonympha.

INTRODUCTION endomicrobes found in the hindgut of termites play a major role in cellulose digestion and manufacture cellulases that Heterotermes indicola (Blattodea: Rhinotermitidae) is a break down cellulose into acetate, propionate and butyrate. lower termite which harbor entozoic flagellates for cellulose They also fix nitrogen that gets incorporated into the termite’s digestion thus causing considerable damage to forests and tissues, excrement and secretions (Mathew et al., 2012). wooden materials throughout the world except Antarctica. Recycling of uric acid by the termite gut bacteria is another About 434 flagellate’s species have been identified in the source of nitrogen (Thong-On et al., 2012). With the aid of hindgut of different termite species which belong to one of the these endomicrobes, such small (termites) cause three orders: Trichomonadida, Hypermastigida and damage worth billions to woods and wooden materials. The Oxymonadida (Inoue et al., 2000). During the last two information pertaining to protozoa, found in the termites as decades termite gut flagellates have been classified using symbionts, will be helpful towards designing strategies for the molecular methods, which are considered to be good tools for production of cellulases and control of lower termites via the species identification and for defining evolutionary inferences development of drugs targeting these protozoa. (Yang and Rannala, 2012). Saldarriaga et al. (2011) described Owing to their importance in the termite hindgut, information the morphology and phylogeny of two Pseudotrichonympha about these endomicrobes and their diversity is extremely species i.e. P. hertwigi from Coptotermes testaceus and P. important. The present work was aimed to investigate the paulistana from Heterotermes tenuis. Phylogenetic analysis diversity of flagellated protozoa with a special focus on P. of fifteen species of Pseudotrichonympha was described by grassii, the largest and the most abundant protozoan found in Noda et al. (2007) from different termite species. However, the hindgut of H. indicola. Moreover, to control the he suggested that only one species of Pseudotrichonympha is population rate of termites and to protect the huge wood found in a termite species at one time. Thus the evolutionary damage and economic loss, it is necessary to eliminate the history of Pseudotrichonympha is important not only due to population of gut flagellates which play an important role in its host specificity but as a host of bacterial endosymbionts, cellulose digestion of termites and made symbiotic which account for more than two third of the total bacterial relationship with each other. population in the termite gut (Noda et al., 2007).The

Ashraf, Qureshi, Haseeb, Hayat, Afzal & Khan

MATERIALS AND METHODS SSU rRNA gene. The PCR was performed on five cells. An initial PCR was done by using set of forward and reverse Study area and Collection of termites: Workers of H. primers (Table 1). The reaction mixture consisted of template indicola were collected from the metropolitan areas of cells, dNTP mix (0.2 mM), primers (1µM each), PCR Buffer Islamabad (33.729388°N, 73.093146°W) (Fig. 1) Pakistan (1X), Ex-Taq DNA polymerase (2.5U) and MgCl2 (1.5mM). from March to May 2017. Termites were kept in Petri dishes Amplification was consisted of 35 cycles of 1 min at 92°C, 1 at 30°C and fed on moistened blotting paper for a week prior min at 50°C and1.5 min at 72°C. Agarose gel electrophoresis to the experiment and identified by using key (Akhtar, 1983). of the reaction revealed no bands. Therefore, using the first This helped in clearing debris and wood particles from reaction contents as a template, a nested PCR was performed. flagellates for distinct morphology. 25µl of PCR products was added with the same constituents as used for primary PCR by using the second set of primers for nested PCR (Table 1). The pieces of gel containing the DNA band were excised from the gel and subjected to DNA purification using the Gene JET Gel Extraction Kit (Cat No. K0691, MBI Fermentas). The pGEM-T-Easy vector (Cat No. A1360, Promega) was used to clone the purified products and transformed into DH5α E. coli cells. This was followed by plasmid DNA extraction and DNA sequencing of the cloned PCR product. Phylogenetic analysis: For the phylogenetic investigation, we analyzed our indigenous P. grassii taxon and compared it with that of isolated from the other localities of the world. The details are given in Table 4. The sequence alignment and data matrix construction were done in the software Geneious® version 6.1 (Biomatters Ltd., New Zealand). Neighbor joining Figure 1. Termites collection sites from Islamabad, analysis was performed using the DNA evolution model Pakistan. (Tamura and Nei, 1993) in the Geneious build algorithm. Wagner parsimony (Farris, 1970) and Maximum likelihood Light microscopy: The hindgut contents of termites were (Guindon and Gascuel, 2003) analysis were carried out using opened in a drop of 0.2% normal saline and tinged with gram PAUP (Swofford, 2002) Geneious plugin. Bayesian analysis iodine solution. Flagellates were observed by using Optika was done using MrBayes (Huelsenbeck and Ronquist, 2001) trinocular digital microscope (Optika B-350, Italy). Images Geneious plugin. Finally, based on the above analysis a and videos were captured in real time with a 12 megapixels consensus evolutionary tree was generated. digital CCD camera on a trinocular Optika microscope. Single cell isolation of P. grassii: The gut of termite workers RESULTS was opened in a 5μl of filter-sterilized buffer (0.1 M NaC1, 10mM Na3PO4, pH 6.9) in the cavity slide. From this mixture Flagellates biodiversity: The protozoan fauna isolated from of gut contents and the buffer, 1ml was taken on another hindgut of H. indicola were categorized according to Adl et cavity slide and diluted further by adding 4ml of the buffer. al. (2005). We identified nine species of flagellates belonged This dilution was repeated four times, 1ml of the final diluted to three genera Holomastigotes, Holomastigotoides and contents was used for observations. The flagellates were Pseudotrichonympha. examined under a digital biological microscope. Individual P. Taxonomic classification of flagellates isolated from H. grassii cells, identified on the basis of morphology, were indicola: picked with a micropipette and collected in PCR tubes. Class Mastigophora (Diesing): Member of class PCR amplification and sequencing: The cells were directly Mastigophora usually have one to several flagella, the nucleus used as a template in a PCR to amplify their nearly full length is vesicular with endosomes.

Table 1. Set of primers used for primary and nested PCR. Primer Name Sequence Reference Primary PCR Eukl8 5’-TGAGGATCCMGGTTGATYCTGCC-3’ Ohkuma et al. (2000) Euk1627 5’-CCGAAGCTTACGGGCGGTGTGTRC-3‘ Nested PCR Har-F 5’-GCGCTACCTGGTTGATCCTGCC-3’ Harper et al. (2009) Har-R 5’-TGATCCTTCTGCAGGTTCACCTAC-3’

944 Profile of protists flagellates

Sub Class Zoomastigia: These may be free living or parasitic deeply stained spiral bands which cover most of the body; and their body lacks chromatophores. Ensysment is usually flagella arise from basal granules; bulge may or may not be common. present at posterior end of body; a long axostyle present; body Order Hypermastigida (Grassii and Foa): Flagellates covered with periplast. belonging to the order Hypermastigida have numerous KEY TO SPECIES flagella and complex cytoplasmic organization. KEY TO FAMILY

Holomastigotes campanula: Diagnosis: Campanula shaped body; broadly round at the

KEY TO GENERA anterior end; truncated posteriorly; body covered with flagella in the form of spiral bands; prenuclear zone absent; axostyle long arises near the nucleus and extend towards the posterior end (Fig. 2, Table 2 & 3). Holomastigotes metchnikowi: Diagnosis: Cylindrical body; rounded interiorly; no protoplasmic bulged; Nucleus at the anterior end; chromatin material at the periphery; spirally arranged flagella are present on all over the body; cytoplasm differentiated into ectoplasm Genus Holomastigotes was described by Grassii in 1882 for st and endoplasm (Fig. 2, Table 2 & 3). the 1 time, later on by de Mello in 1927, Kudo in 1947 and Holomastigotes annandalei: Saleem in 1952. Diagnosis: Body oval shaped with broadly round anterior Diagnosis: Nucleus not surrounded by dense protoplasm; end; nucleus anterior in position; chromatin in the form of 2 basal granules are present deep in the cytoplasm and form to 3 patches; axostyle present; flagella of uniform size present

Table 2. Synonym, type host and additional host of the representative species reported from literature. Name of species Synonym Type host Add Host Holomatigotes Leidya campanula (De Mello, 1927) Heterotermes indicola Heterotermes indicola campanula Holomastigotiodes campanula (De Mello, 1927) (Portergaise) (Islamabad) Holomastigotes campanula (Saleem, 1952) Holomatigotes Leidya metchnikowi (Franca, 1919) Heterotermes indicola Heterotermes indicola metchnikowi (Portergaise) (Islamabad) Holomastigotoides gigas (De Mello, 1927) Holomatigotes Heterotermes indicola Heterotermes indicola annandalei (Lahore) (Islamabad) Holomastigotoides Leidya metchnikowi (Franca, 1919) Coptotermes heimi Heterotermes indicola metchnikowi Holomastigotoides metchnikowi (De Mello, 1927) (Islamabad) Holomastigotoides Leidya kempi (De Mello, 1919) Heterotermes indicola Kempi - (Islamabad) Holomastigotoides Holomastigotoides hemigynum (Grassii, 1917) Coptotermes lecteus Lecuotermes indicola (Brazil) hemigynum Holomastigotoides hemigynum (De Mello, 1927) (Austerlia) Heterotermes indicola (Portergaise) Holomastigotoides hemigynum (Saleem, 1952) Coptotermes heimi (Lahore) Leidya annadalei (De Mello, 1919) Heterotermes indicola (Islamabad) Holomastigotoides Holomastigotoides hartmanni (Koidzumi, 1921) Heterotermes indicola Heterotermes indicola hartmanni (Portergaise) (Islamabad) Holomastigotoides H. hartmanni (De Mello, 1927) Heterotermes indicola Holomastigotoides hartmanni (Saleem, 1952) (Lahore)

Pseudotrichonympha Coptotermes formosanus Heterotermes indicola grassii - Coptotermes heimi

945 Ashraf, Qureshi, Haseeb, Hayat, Afzal & Khan

Table 3. Sample T-test for the morphometric diversity of flagellates isolated from Heterotermes indicola. Species Parameters N O.R x̅ S.D S.E 95% CI Holomastigotoides Body Length 25 17.45-132.0 65.46 15.46 8.58 47.36-83.56 kempi Body width 25 11.9-108.00 51.95 12.65 7.56 36.00-67.89 Nucleus Diameter 25 8.38-15.00 10.45 2.05 0.48 9.43-11.47 Flagella Length 25 4.76-96.00 39.86 26.76 6.31 26.56-51.17 Bulge Length 25 4.76-62.70 32.64 19.07 4.50 23.15-42.12 Bulge width 25 9.52-96.00 39.61 25.99 6.13 26.68-52.53 Holomastigotoides Body Length 24 20.04-692.0 122.4 34.80 11.40 36.7-189.80 metchnikowi Body width 24 19.84-618.0 123.8 40.80 10.60 38.3-199.80 Nucleus Diameter 24 4.05-83.00 20.25 4.40 2.80 7.67-18.46 Flagella Length 24 2.00-71.38 15.07 7.50 2.50 6.13-14.45 Holomastigotoides Body Length 12 15.08-74.26 47.90 22.76 8.05 28.87-66.92 hartmanni Body width 12 9.28-22.00 16.82 5.23 1.85 12.44-21.19 Nucleus Diameter 12 3.00-23.80 3.50 1.55 0.55 2.20-4.800 Flagella Length 12 2.19-8.530 5.31 2.17 0.76 3.49-7.120 Holomastigotoides Body Length 18 14.29-43.00 26.91 9.87 2.33 28.87-66.92 hemigynum Body width 18 9.81-24.09 16.86 3.89 0.91 19.02-44.54 Nucleus Diameter 18 2.96-6.040 3.45 0.94 0.22 2.20-3.910 Flagella Length 18 7.14-11.90 9.33 1.43 0.33 8.62-10.04 Holomastigotoides Body Length 20 21.23-72.00 49.65 18.70 4.18 40.90-58.40 Koidzumi Body width 20 18.88-54.00 28.96 12.36 2.76 23.17-34.74 Nucleus Diameter 20 2.50-10.08 6.23 1.74 0.39 5.41-7.050 Flagella Length 20 1.98-8.200 3.70 1.40 0.31 3.04-4.350 Axostyle length 20 3.00-12.00 6.95 3.45 0.77 5.33-8.570 Holomastigotes Body Length 15 14.78-62.21 36.25 19.06 6.70 20.31-52.19 annandalei Body width 15 7.65-58.00 29.25 17.68 6.25 14.47-44.03 Nucleus Diameter 15 3.10-15.39 3.62 1.40 0.49 2.44-4.800 Flagella Length 15 1.73-6.350 3.50 1.43 0.50 2.29-4.700 Bulge Length 15 3.40-7.070 7.38 3.66 1.29 4.31-10.44 Bulge width 15 7.89-22.07 20.38 12.49 4.42 9.93-30.82 Holomastigotes Body Length 22 16.38-142.0 81.68 43.45 9.26 62.42-100.9 metchnikowi Body width 22 13.00-64.26 49.20 15.85 3.38 42.17-56.22 Nucleus Diameter 22 3.45-14.00 9.06 2.38 0.50 8.00-10.11 Flagella Length 22 2.36-13.00 7.92 3.23 0.69 6.49-9.350 Holomastigotes Body Length 22 118.0-239.00 157.92 3.23 0.69 6.49-9.350 campanula Body width 22 9.09-41.00 20.18 10.32 2.20 15.61-24.76 Nucleus Diameter 22 2.90-9.450 5.02 1.14 0.24 4.51-5.530 Flagella Length 22 2.00-5.000 2.34 0.64 0.13 2.05-5.620 Pseudotrichonympha Body Length 22 9.04-689.0 184.90 39.70 7.90 89.7-250.10 grassii Body width 22 15.30-610.0 158.70 34.36 7.34 76.1-187.30 Nucleus Diameter 22 2.38-7.700 5.07 1.35 0.28 4.47-5.670 Flagella Length 22 3.57-8.400 5.44 1.67 0.35 4.75-6.240 Length of 22 3.23-8.000 4.13 0.77 0.16 3.79-4.480 Centroblepharoplast in the form of dextral spiral bands on all over the body; end of body, may be smooth or covered with flagella; long protoplasmic groove is present at posterior end; No axostyle present; body covered with periplast. distinction of ectoplasm or endoplasm (Fig. 2, Table 2 & 3). Genus Holomastigotoides: Holomastigotoides was described by Grassii and Foa (1911) for the 1st time, later on by de Mello (1927), Kudo (1947) and Saleem (1952). Diagnosis: Prenuclear zone present, consists of dense cytoplasm; Basal granules found deep in the cytoplasm, in the form of deeply stained spiral bands, cover most of the body; flagella arise from basal granules extend from anterior to posterior end; bulge may or may not be present at the posterior

946 Profile of protists flagellates

Figure 2. Different microscopic views of (i) Holomastigotes campanula (ii) Holomastigotes metchnikowi (iii) Holomastigotes annandalei at 100X; NU (Nucleus); PRPL (Protoplasm); PRPL GRV (Protoplasmic groove); SPR BND (Spiral bands); Figure 3. Different microscopic views of (i) CHR (Chromatin); FLG (Flagella); W PART Holomastigotoides metchnikowi (ii) (Wood particles). Holomastigotoides koidzumi (iii) Holomastigotoides kempi at 100X; NU (Nucleus); KEY TO SPECIES PRPL (Protoplasm); PRPL GRV (Protoplasmic Holomastigotoides metchnikowi: groove); SPR BND (Spiral bands); CHR Diagnosis: Body bottle shaped; body breadth 1/3rd to 1/4th of (Chromatin); AX (Axostyle); FLG (Flagella); W the entire length and is uniform till the neck; protoplasmic PART (Wood particles). P. NU. ZN (Pre-nuclear bulge absent; distinguishable ectoplasm and endoplasm; zone); GRV (Groove); PRPLSM BG (Protoplasmic flagella arise from basal granules in cytoplasm and are bulge). arranged in spiral rows; nucleus lies slightly anterior to the middle of the body; chromatin material aggregated to the Holomastigotoides kempi: periphery of the nucleus; axostyle present and projects Diagnosis: Oval shaped body with its anterior part slightly downward from the nucleus (Fig. 3, Table 2 & 3). drawn out forming a nipple shape; no differentiation of Holomastigotoides koidzumi: cytoplasm into ectoplasm and endoplasm; broad protoplasmic Diagnosis: Body cone-shaped anteriorly; truncated bulge present at posterior end of body; flagella cover whole posteriorly; nucleus round and anterior in position; no the body; nucleus round in shape; distinct nuclear membrane differentiation of cytoplasm into ectoplasm and endoplasm; present; chromatin material arranged in the form of groups or prenuclear zone distinct from body wall is arranged into spiral strands; 2 to 3 nucleoli generally visible, their number varies rows; long flagella are present and flagella bands are whorled with individuals; axostyle posterior to the nucleus and three times around the body; nucleus surrounds axostyle protrudes downwards; prenuclear zone present (Fig. 3, which extends to posterior (Fig. 3, Table 2 & 3). Table 2 & 3).

947 Ashraf, Qureshi, Haseeb, Hayat, Afzal & Khan

Holomastigotoides hemigynum: Diagnosis: spindle shaped body divided into two distinct Diagnosis: Body cone-shaped; no differentiation of parts; bell and body proper; bell at the anterior end of the protoplasm into ecto and endoplasm; round nucleus has 1 to centroblepharoplast is barrel-shaped and covered by a cap; 2 nucleoli and covered by a nuclear membrane; chromatin spiral rows of flagella present on the whole body; cytoplasm granules arranged in 2 to 3 groups; axostyle projects divided into ectoplasm and endoplasm. downwards into the naked protoplasmic bulge from the Pseudotrichonympha grassii: posterior end of nucleus (Fig. 4, Table 2 & 3). Diagnosis: Spindle-shaped body; divided into head, neck and body proper; flagella arranged in the form of dextral spiral rows completely cover the body; head composed of a bell covered by a transparent hyaline cap; upper margin of the cap lies close to the bell and lower margin folded and devoid of flagella; neck also called the centroblepharoplast, tube like anteriorly and has varying thickness; centroblepharoplast composed of basal granules lying close to each other, which appear to constitute a single structure; neck flagella long, active, in the form of three series and perform jerky movements; flagella of Series 1 arise from the bell; their length increase from top to bottom and showed strong jerky movements. Flagella that root from the centroblepharoplast are referred to as flagella of series 2 and they are longer than the flagella of series 1; Flagella of series 3 arise from the posterior region of the neck, are larger nucleus than the other two series and perform very active jerky movements during locomotion; round or oval in shape; has two nucleoli; chromatin material is evenly distributed or scattered randomly; nucleus has no definite position; it can be at the anterior end, the middle part, or the posterior end of the body (Fig. 5, Table 2 & 3).

Figure 4. Different microscopic views of (i) Holomastigotoides hemigynum and (ii) Holomastigotoides hartmani at 100X; NU (Nucleus); PRPL (Protoplasm); SPR BND (Spiral bands); CHR (Chromatin); AX (Axostyle); FLG (Flagella); W PART (Wood particles) STR CL (Steriocilia); P. NU. ZN (Pre-nuclear zone).

Holomastigotoides hartmanni: Diagnosis: Body pear-shaped with elongated and slightly pointed anterior part; most of the body covered with periplast which helps in permanency of body shape; posterior part of the body without periplast, no spiral bands are present; steriocilia are attached; the steriocilia are different from cilia because of their mode of attachment and are devoid of basal granules; flagella arise from basal granules that lies in the cytoplasm; nucleus lies near the anterior end; have 1to3 nucleoli (Fig. 4, Table 2 & 3). Figure 5. Different microscopic views of GENUS Pseudotrichonympha grassii at 100X; CP (Cap); Pseudotrichonympha: The genus Pseudotrichonympha is BL (Bell.); CNTRBLP (Centroblepharoplast); described by Grassii in 1917 for the first time as PLSM ECT (Ectoplasm); PLSM END Trichonympha, then as Pseudotrichonympha by de Mello in (Endoplasm); W PART (Wood particles); NU 1927, Saleem in 1952 and Kudo in 1966. (Nucleus); BAC (Bacteroidales); FLG (Flagella).

948 Profile of protists flagellates

Table 4. Accession number of Pseudotrichonympha grassii from NCBI used for constructing a phylogenetic tree. Taxon NCBI Gene Bank accession Country of origin In group Pseudotrichonympha grassii JX649947 Pakistan Pseudotrichonympha grassii AB262511 Japan Pseudotrichonympha grassii AB262486 China Pseudotrichonympha grassii AB032211 Japan Pseudotrichonympha sp. AB262487 Malaysia Pseudotrichonympha sp. AB262488 Laos Pseudotrichonympha sp. AB262491 Brazil Pseudotrichonympha hertwigi HQ683706 Canada Pseudotrichonympha paulistana HQ683705 Canada Out group Barbulanympha ufalula AB443595 Japan

PCR amplification of SSU rRNA: Two sets of primers (Table 1) were used for amplification of the SSU rRNA gene. The amplification was confirmed by performing gel electrophoresis assay and the results revealed a DNA band of the expected size of 1.4 KB (Fig. 6).

1.4 kb 1.5 kb 1.0 kb

Figure 7. Molecular phylogenetic tree of P. grassii (NJ, Neighbor Joining; BP, Bayesian Probability; PAR, Parsimony; ML, Maximum Likelihood; for the Figure 6. PCR amplified product of SSU rRNA of P. details of taxa used. grassii. DISCUSSION Phylogenetic analysis of P. grassii: BLAST analysis of the DNA sequence of the cloned PCR product performed on In the present studies, the nine species of flagellates belonging NCBI’s website (http://blast.ncbi.nlm.nih.gov/) confirmed to three genera viz, Holomastigotes (H. campanula, H. that the PCR product was P. grassii SSU rRNA gene. The annandalei and H. metchnikowi), Holomastigotoides (H. phylogenetic tree shows that P. grassii isolated from Pakistan hemigynum, H. hartmanni, H. kempi, H. koidzumi and H. closely relates to the P. grassii isolated from Japan. Bayesian metchnikowi) and Pseudo trichonympha (P. grassii) were probability together with bootstrap values for Niebuhr identified from H. indicola morphologically. In addition, of Joining, Parsimony and Maximum Likelihood analysis P. grassii was identified on a molecular basis analyzed strongly supported our phylogenetic tree and its clades phylogenetically. Studies on the relative abundance of some (Fig. 7, Table 4). The accession JX649947 was obtained after species of protists in the hindgut of H. indicola demonstrated the submission of sequences in Gene Bank. that P. grassii is the largest and the most abundant species found in H. indicola. The comparison of our P. grassii isolates

949 Ashraf, Qureshi, Haseeb, Hayat, Afzal & Khan with the P. grassii isolate in other parts of the world, the REFERENCES results of our molecular phylogenetics are in agreement with those of Noda et al. (2005). Evolutionary tree (Fig. 7) depicts Adl, S.M., A.G. Simpson, M.A. Farmer, R.A. Andersen, O.R. that Pakistani P. grassii isolate was closely related to the Anderson, J.R. Barta, S.S. Bowser, G.U.Y. Brugerolle, Japanese P. grassii isolate and falls in the Asian clade. R.A. Fensome, S. Fredericq and T.Y. James. 2005. The In the termite hindgut P. grassii is found replete with wood new higher level classification of eukaryotes with particles it is suggested that wood digestion largely depends emphasis on the taxonomy of protists. J. Eukaryot. upon this comparatively large flagellate. The P. grassii Microbiol. 52:339-451. digests lignocellulose in the wood which is the major component of cellulose and provide digested food material to Akhtar, M.S. 1983. Wood destroying termites (Isoptera) of the termites. Thus P. grassii may alone be sufficient to fulfill Pakistan: Key to the most important species, their the entire nutritional needs of the termite and dominates over distribution and pattern of attack. Mater. Org. 18:277- the significance of other flagellate species. Therefore, 291. antiprotozoal drugs against P. grassii may prove a valuable De Mello, I.F. 1919. Contributiona I’etude dela faune tool for termite eradication. Pseudotrichonympha occurs parasitaaire d’ Hodotermes vorum koening de coim almost exclusively in Rhinotermitidae except for the genus batore (lnde Ahglaise) Ibid. Reticulitermes closely related to Heterotermes and De Mello, I.F. 1927. Revision des trichonympides du Coptotermes (Inward et al., 2007: Lo et al., 2004). It is the Leucotermes indicola Wasmann, Arquivos da Escola only parabasalian symbiont identified in the genera medico. Ser Cirugica de nova Goa 1:1-28. 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Keeling. 2009. The inadequacy of morphology for species and genus delineation in microbial eukaryotes: an Conclusion: The endomicrobes found in the hindgut of example from the parabasalian termite symbiont termites play a major role in cellulose digestion and Coronympha. PLoS One 4:e6577. manufacture cellulases that break down cellulose into acetate, Huelsenbeck, J.P. and F. Ronquist. 2001. MRBAYES: propionate and butyrate. On the other hand, with the aid of Bayesian inference of phylogenetic trees. Bioinformatics these endomicrobes such small insects cause millions of 17:754-755. damages to woods and wooden materials. Thus, by reviewing Inoue, T., O. Kitade, T. Yoshimura and I. Yamaoka. 2000. morphology and characteristics of symbiotic flagellates Symbiotic associations with protists. In: T. Abe, D.E. residing in the hindgut of H. indicola it was proposed that Bignell, M. 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950 Profile of protists flagellates

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